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PubMed2005

Standard line slopes as a measure of a relative matrix effect in quantitative HPLC-MS bioanalysis.

A simple experimental approach for studying and identifying the relative matrix effect (for example "plasma-to-plasma" and/or "urine-to-urine") in quantitative analyses by HPLC-MS/MS is described. Using as a database a large number of examples of methods developed in recent years in our laboratories, the relationship between the precision of standard line slopes constructed in five different lots of a biofluid (for example plasma) and the reliability of determination of concentration of an analyte in a particular plasma lot (or subject) was examined. In addition, the precision of standard line slopes was compared when stable isotope-labeled analytes versus analogs were used as internal standards (IS). Also, in some cases, a direct comparison of standard line slopes was made when different HPLC-MS interfaces (APCI versus ESI) were used for the assay of the same compound, using the same IS and the same sample preparation and chromatographic separation conditions. In selected cases, the precision of standard line slopes in five different lots of a biofluid was compared with precision values determined five times in a single lot. The results of these studies indicated that the variability of standard line slopes in different lots of a biofluid [precision of standard line slopes expressed as coefficient of variation, CV (%)] may serve as a good indicator of a relative matrix effect and, it is suggested, this precision value should not exceed 3-4% for the method to be considered reliable and free from the relative matrix effect liability. Based on the results presented, in order to assess the relative matrix effect in bioanalytical methods, it is recommended to perform assay precision and accuracy determination in five different lots of a biofluid, instead of repeat (n=5) analysis in the same, single biofluid lot, calculate standard line slopes and precision of these slopes, and to use <3-4% slope precision value as a guide for method applicability to support clinical studies. It was also demonstrated that when stable isotope-labeled analytes were used as internal standards, the precision of standard line slopes in five different lots of a biofluid was </=2.4% irrespective of the HPLC-MS interface utilized. This clearly indicated that, in all cases studied, the use of stable isotope-labeled IS eliminated relative matrix effect. Also, the utilization of the APCI interface instead of ESI led to the elimination of the relative matrix effect in all cases studied. When the precision of standard line slope values exceeds the 3-4% limit, the method may require improvements (a more efficient chromatography, a more selective extraction, a stable isotope-labeled IS instead of an analog as an IS, and/or a change in the HPLC-MS interface) to eliminate the relative matrix effect and to improve assay selectivity.

Body Fluids↗
PubMedDate not supplied

Exposure to cadmium from foods, estimated by analysis and calculation--comparison of the methods.

Cadmium intake of 40 middle-aged Finnish men was determined both by calculation based on computer files and by analysis of duplicate portions. A significant difference was observed between intake estimates of the two methods. The average calculated cadmium intake was 15.8 micrograms/day compared to the 8.2 micrograms/day obtained by analysis. Factors causing the difference between the methods and affecting the comparability are: (i) expression of concentrations below the detection limit; (ii) the calculation is based on the analysis of foods being only washed and peeled whereas duplicate portions are normally processed; (iii) when duplicate portions are prepared using non-representative lots or brands of food, their cadmium content differs from average values used in calculation; (iv) the level of cadmium content in duplicate portion samples is very low affecting the accuracy of the results. The analytical level of cadmium during the analysis of duplicate portions was somewhat lower as compared to the analysis on which the food composition file and the calculation is based. It seems that the complicating factors identified in the present study potentially affect the reliability and comparability of studies of trace element intakes. Therefore one has to be careful when comparing intake estimates derived independently or by different methods.

Cadmium↗
PubMed2005

Measurement of stable isotopic enrichment and concentration of long-chain fatty acyl-carnitines in tissue by HPLC-MS.

We have developed a new method for the simultaneous measurements of stable isotopic tracer enrichments and concentrations of individual long-chain fatty acyl-carnitines in muscle tissue using ion-pairing high-performance liquid chromatography-electrospray ionization quadrupole mass spectrometry in the selected ion monitoring (SIM) mode. Long-chain fatty acyl-carnitines were extracted from frozen muscle tissue samples by acetonitrile/methanol. Baseline separation was achieved by reverse-phase HPLC in the presence of the volatile ion-pairing reagent heptafluorobutyric acid. The SIM capability of a single quadrupole mass analyzer allows further separation of the ions of interest from the sample matrixes, providing very clean total and selected ion chromatograms that can be used to calculate the stable isotopic tracer enrichment and concentration of long-chain fatty acyl-carnitines in a single analysis. The combination of these two separation techniques greatly simplifies the sample preparation procedure and increases the detection sensitivity. Applying this protocol to biological muscle samples proves it to be a very sensitive, accurate, and precise analytical tool.

Animals↗
PubMed1991

[Some criteria for the planning and evaluation of the ruggedness test of HPLC methods].

Ruggedness is a measure of the reproducibility of the individual test results when the procedure is used repeatedly to determine the same homogeneous sample in a variety of specified experimental conditions. Ruggedness testing can be divided into two major groups: Type-A, when the influence of the changed environmental conditions (different equipment, analysts, etc.) on the analytical performance parameters and results are tested, and Type-B, when the effect of variation in the experimental conditions are checked. In the latter case, Type-B group can be further classified into two major subgroups: ruggedness testing of sample preparation and chromatographic separation. Checking sample preparation for ruggedness testing includes the investigation of various sample preparation procedures, such as: liquid-liquid extraction, solid phase extraction, direct sample introduction and derivative formation. The investigation of column-to-column variability, changes in the instrumental and experimental conditions belongs to the ruggedness testing of chromatographic separation. In this paper the basic principles of selection for experimental variables for various ruggedness tests are discussed introducing a new possibility to design multivariable-multicriteria system, and the evaluation of a single-variable-single-criterion system, as well. In general, from the work introduced in this paper, it can be concluded that when new analytical methods are developed, it is quite important to be aware the sensitivity of the method to variations in the specified conditions. For adequate performance of ruggedness testing: a) the factors (variables) which have significant influence on the chromatographic results can be firstly selected, b) maximum acceptable deviations from the prespecified experimental conditions can be decided, and c) the acceptable deviations from the values of the prespecified analytical performance parameters can be defined.

Chromatography, High Pressure Liquid↗
PubMed2006

Microbiological and physicochemical stability of fentanyl and sufentanil solutions for patient-controlled delivery systems.

The aim of this study was to assess the microbiological and physicochemical stability of opioid solutions containing fentanyl or sufentanil and thereby determine the feasibility of extending the expiration dates after mixing. Five systems containing fentanyl or sufentanil solutions at 50 microg/mL in portable patient-controlled analgesia (PCA) systems were filled and stored at room temperature for 14 days. They were sampled immediately after preparation, at day 3, and each day of the following weeks. Microbiological stability was assessed by performing sterility tests. The physicochemical study was performed by determining aspect, pH, and osmolality evolution. All samples were tested for appearance, change in color, and loss of concentration using an analytical method. There was no significant change in pH and osmolality values of any solutions. No precipitation or change in color was observed in any of the sample solutions. There was no significant loss of fentanyl or sufentanil over 14 days (4.3% and 4.1%, respectively). This study indicates that both drug solutions in the PCA systems are stable for a minimum of 14 days at room temperature.

Analgesia, Patient-Controlled↗
PubMed2000

High-performance liquid chromatography-diode array detection of trichloroethene and aromatic and aliphatic anionic surfactants used for surfactant-enhanced aquifer remediation.

A method utilizing direct aqueous injection with high-performance liquid chromatography and diode array detection (HPLC-DAD) is presented for the quantitation determination of trichloroethene (TCE) in the presence of anionic surfactants that are used to enhance the recovery of dense non-aqueous phase liquids from contaminated groundwater aquifers. The anionic surfactants investigated in this study including alkyl diphenyloxide disulfonate (Dowfax 8390) and dihexylsulfosuccinate (Aerosol MA 80-1) are used to enhance the solubility, and hence recovery, of TCE. In this type of environmental engineering application, the levels of surfactants and TCE encountered are very high (part per million to part per thousand). The anionic surfactants and TCE are quantitatively determined by direct aqueous injection onto a reversed-phase HPLC column with diode array detection. The quantitation limits of the method obtained using 100 microl injections are 0.1 mg/l for alkyl diphenyloxide disulfonates, 20 mg/l for dihexylsulfosuccinate, and 0.05 mg/l for TCE. This approach is advantageous over using gas chromatography for TCE and HPLC for the surfactants because the use of a single analytical instrument reduces sample preparation and analysis times, which increases sample throughput.

Chromatography, High Pressure Liquid↗
PubMed2002

Application of liquid chromatography-mass spectrometry to the quantification of bisphenol A in human semen.

The potential risks to human health and reproduction from the xenoestrogen bisphenol A (BPA) have not been well established. This is due in part to the absence of accurate analytical methods to quantify BPA in biological samples. In this study we establish an accurate, sensitive and selective analytical method for the quantification of BPA in human semen. To quantify BPA we compared the techniques of liquid chromatography-mass spectrometry (LC-MS) and enzyme-linked immunosorbent assay (ELISA). In addition we have taken steps to eliminate BPA contamination during sample extraction and preparation. Results show that the ELISA method gives an over-estimate of BPA concentration, which may be due, at least in part, to non-specific interactions with the BPA-antibodies. LC-MS gave much more accurate results and proved to be more sensitive with a detection limit of 0.5 ng ml(-1) compared to 2.0 ng ml(-1) by ELISA.

Benzhydryl Compounds↗
PubMed2002

Analysis of quartz by FT-IR in air samples of construction dust.

The construction industry is reported to have some of the highest exposures to silica-containing dust. With the designation of crystalline silica as a group I human carcinogen by the International Agency for Research on Cancer (IARC), there exists a need for an analytical method to accurately quantify low levels of quartz. A method is described that uses FT-IR for quartz analysis of personal air samples collected from heavy and highway construction sites using 4-stage personal impactors. Sample filters were ashed and 13-mm or 5-mm pellets were prepared. Absorbance spectra were collected using FT-IR at resolution of 1 cm(-1) and 64 scans per spectrum. Two spectra were collected per sample using the appropriate background spectrum subtraction. Spectral manipulations such as Fourier self-deconvolution and derivatizations were performed to improve quantification. Peak height for quartz was measured at 798 cm(-1) for quantitative analysis. The estimated limit of detection for the 5-mm pellets was 1.3 microg. Recoveries of Min-U-Sil 5 spikes showed an average of > or = 94 percent for the two pellet types. The coefficient of variation of the 5-mm pellet was 9 percent at 6 microg quartz load, and 7 percent at 62 microg load. Interferences from clay, amorphous silica, concrete, calcite, and kaolinite were investigated, these being the more likely sources of interferences in construction environment. Spikes of mixtures of amorphous silica or kaolinite with Min-U-Sil 5 showed both contaminants introduced, on average, a positive error of < 5 microg with average recoveries of 106 percent and 111 percent, respectively. Spikes of mixtures of clay or concrete with Min-U-Sil 5 showed overall average recovery of 100 percent and 90 percent, respectively, after accounting for the presence of quartz in clay and concrete. This method can quantify low levels of quartz with reasonable accuracy in the face of common contaminants found in the construction industry.

Air Pollutants↗
PubMed1980

Gas chromatographic methods for the determination of antiepileptic drugs: a systematic review.

Despite the availability of a variety of methods for determining the concentrations of antiepileptic drugs in body fluids--including thin-layer chromatography, high pressure liquid chromatography, radioimmunoassay, etc.--gas chromatography remains perhaps the routinely used tool for therapeutic drug monitoring in this area. As a result of the need to improve precision and reproducibility, as well as to reduce costs, among other factors, there has been a continuous technical improvement in the technique. And in the last 10 years, more than 100 papers have described gas chromatography for the determination of antiepileptic drugs. Needless to say, only a small percentage of these publications described methods of a significantly new character. In order to facilitate the task, of comparing methods for the purpose of choosing a technique suitable to a specific need, we have reviewed the analytic parameters described in the 114 publications of the last decade. These parameters include the following: (1) drugs determined, (2) internal standard, (3) amount of sample, (4) extraction procedure, (5) preparation of derivatives, (6) sample injection, (7) detection, (8) column support and temperature, and (9) stationary phase. We present these data in a comprehensive tabular form together with a number of comments concerning specific techniques.

Anticonvulsants↗
PubMed2002

Improvement of "hit-to-lead" optimization by integration of in vitro HTS experimental models for early determination of pharmacokinetic properties.

Development of predictive in vitro surrogate methods for traditional approaches assessing bioavailability and pharmacokinetics of lead compounds must be made to both keep pace with high-throughput (HT) lead identification and to mitigate the high costs associated with progression of compounds with poor chances of developmental success. Indeed opportunities for improvement still exist in the lead optimization phase versus the lead identification phase, where HT methodologies have been nearly optimized. Review of examples, limitations, and development of high-throughput microtiterplate-based assays for evaluating metabolic liabilities, such as in vitro radiometric and fluorometric assays for inhibition of cytochrome p450 (CYP) activity, determination of stability of a compound in liver microsomes, or cloned CYPs coupled to reconstituting systems are described. Parallel approaches to improve speed, resolution, sample preparation, as well as data analysis using LC/MS and LC/MS/MS approaches and technologies to assess compound integrity and biotransformation by automation and multiplexing are also discussed. Realization of the benefits in automation of cell-based models for determining drug permeability to predict drug absorption are still hampered by bottlenecks in analytical analysis of compounds. The implementation and limitations of surrogate physiochemical methods for passive adsorption such as immobilized artificial membranes (IAM) and parallel artificial membrane permeation assays (PAMPA), and compound solubility by laser nephelometry are reviewed as well. Additionally, data from a high-throughput 96-well equilibrium dialysis device, showing good correlation to classical methods, is presented. Finally, the impact of improvements in these downstream bottlenecks in lead optimization and preclinical drug discovery are discussed in this review.

Animals↗
PubMed1990

Liquid chromatographic assay and pharmacokinetics of halazepam and its metabolite in humans.

A reversed-phase high-performance liquid chromatographic method is described for simultaneous quantification of halazepam and its major active metabolite, nordiazepam, in plasma. The method uses a solid-phase extraction procedure to prepare plasma samples. After extraction, the methanolic extract is evaporated, and the residue is then reconstituted in a small volume of mobile phase (a 40:60, v/v, mixture of 0.02 M phosphate buffer, pH 4.0, and methanol) and chromatographed. The total chromatography time for a single sample is approximately 10 min. A sensitivity of 1 ng/mL for halazepam and nordiazepam is attained when 1 mL of plasma is extracted. Analytical recovery of halazepam and nordiazepam added to the plasma ranged from 89 to 96%. The maximum within-day and day-to-day coefficients of variation for each compound at the concentration range of 2 to 100 ng/mL were 8.7 and 10.3%, respectively. Suitability of the method was assessed in a preliminary pharmacokinetic study in which three subjects were given a single 20-mg oral dose of halazepam following an overnight fast. It appeared from our kinetic analysis that halazepam is a drug with a fairly rapid absorption phase that is followed by a slow elimination phase. Mean oral plasma clearance of halazepam was 24 L/h. The mean apparent elimination half-life of nordiazepam (45.22 h) is considerably longer than that of halazepam (21.15 h).

Administration, Oral↗
PubMed2002

Isotope ratios of trace elements in samples from human nutrition studies determined by TIMS and ICP-MS: precision and accuracy compared.

Stable isotopes are used with increasing frequency to trace the metabolic fate of minerals in human nutrition studies. The precision of the analytical methods used must be sufficient to permit reliable measurement of low enrichments and the accuracy should permit comparisons between studies. Two methods most frequently used today are thermal ionization mass spectrometry (TIMS) and inductively coupled plasma mass spectrometry (ICP-MS). This study was conducted to compare the two methods. Multiple natural samples of copper, zinc, molybdenum, and magnesium were analyzed by both methods to compare their internal and external precision. Samples with a range of isotopic enrichments that were collected from human studies or prepared from standards were analyzed to compare their accuracy. TIMS was more precise and accurate than ICP-MS. However, the cost, ease, and speed of analysis were better for ICP-MS. Therefore, for most purposes, ICP-MS is the method of choice, but when the highest degrees of precision and accuracy are required and when enrichments are very low, TIMS is the method of choice.

Chemistry Techniques, Analytical↗
PubMed2001

Validation of a simple and sensitive gas chromatographic method for the analysis of tri-n-butyl phosphate from virally inactivated human immunoglobulin.

Tri-n-butyl phosphate (TnBP), a solvent used in combination with Triton X-100 to inactivate lipid-enveloped viruses from immunoglobulin purified from human plasma is routinely measured in our laboratories by gas chromatography-flame ionization dectection (GC-FID) after extraction with C-18. We modified our present assay by extracting the analyte into hexane prior to measurement by GC-FID. We also found that the addition of a small volume of ethanol to the organic layer facilitates the extraction process by breaking the resulting emulsion formation caused by the hexane addition. The sample preparation and subsequent assay were fully validated in our laboratory. The process time for each sample is less than 2 min, a 15-fold improvement over solid-phase extraction techniques that were previously used in our laboratories. The recovery of TnBP in immunoglobulin using this newer method approximates 100%. The limit of quantitation (LOQ) was found to be 2 microg/ml or 2 ng per injection. The linear dynamic range of the assay is reported to be from the LOQ up to 50 microg/ml. The method is simple, relatively inexpensive and rapid. In addition, validation of the method demonstrates that it is accurate, precise, rugged and robust as demonstrated by reproducibility between analysts, instruments, laboratories, and columns. Finally, no problems were observed with regard to sample carryover.

Chromatography, Gas↗
PubMed1996

High-performance liquid chromatographic evaluation of biogenic amines in foods. An analysis of different methods of sample preparation in relation to food characteristics.

Biogenic amines are compounds formed by amino acid decarboxylation in fermented foods. Most of the methods for amine determination involve acid extraction followed by a liquid-liquid purification step. The different parameters which can influence amine recoveries are considered; experience with different foods such as cheese, fish and meat preserves are reported and for each of them the optimized analytical procedure is described. Data concerning recovery and repeatability of the method are also reported and the various factors that influence amine extraction are discussed. The possibility of applying direct derivatization without any other purification step is also considered.

Biogenic Amines↗
PubMed2004

Stability of lisinopril in two liquid dosage forms.

BACKGROUND: Lisinopril is used in pediatric patients with hypertension. It is not commercially available as a liquid. Little is known about the stability of lisinopril in extemporaneously prepared liquid dosage forms. OBJECTIVE: To determine the stability of lisinopril in 2 oral suspensions stored at 4 and 25 degrees C in plastic prescription bottles. METHODS: Five bottles contained methylcellulose 1%:simple syrup NF (1:13) and the other 5 bottles had Ora Plus-Ora Sweet (1:1) at a lisinopril concentration of 1 mg/mL. Three samples were collected from each bottle at 0, 7, 14, 28, 42, 56, 70, and 91 days and analyzed by stability-indicating HPLC analytical method (n = 15). RESULTS: At 4 degrees C, the mean +/- SD concentration of lisinopril remained >95.1 +/- 1.8% of the initial concentration in the methylcellulose formulation and 95.1 +/- 3.2% of the initial concentration in the Ora Plus-Ora Sweet formulation throughout the 91-day study period. At 25 degrees C, the mean concentration of lisinopril remained >92.4 +/- 2.2% of the initial concentration in the methylcellulose formulation for 8 weeks and 95.8 +/- 2.3% of the initial concentration in the Ora Plus-Ora Sweet formulation throughout the 91-day study period. No changes in physical appearance in any samples were seen during this period. CONCLUSIONS: Lisinopril can be prepared in either of 2 liquid dosage forms and stored for at least 13 weeks under refrigeration and 8 weeks at room temperature.

Administration, Oral↗
PubMed2005

Optimization of the preparative separation of a chiral pharmaceutical intermediate by high performance liquid chromatography.

The prediction of optimal conditions of the preparative HPLC separation of the enantiomers of a pharmaceutical intermediate was accomplished by employing analytical chromatographic data, i.e. sample injections at low concentrations. Various temperatures and mobile phase conditions were studied. It was assumed that the sample loadability of the stationary phase is constant for a constant value of the separation factor and different mobile phase conditions and temperatures. Using this assumption, possible production rates can be compared for different method conditions. Overloading experiments were carried out to verify that the procedure employed is adequate. It was found that the optimization approach used, changing the mobile phase composition and temperature to achieve the shortest cycle time while keeping the separation factor constant, could be applied to improve the production rate of the separation.

Chromatography, High Pressure Liquid↗
PubMed2005

Separation of proteins with a molecular mass difference of 2 kDa utilizing preparative double-inverted gradient polyacrylamide gel electrophoresis under nonreducing conditions: application to the isolation of 24 kDa human growth hormone.

A method for separating proteins with a molecular mass difference of 2 kDa using SDS-PAGE under nonreducing conditions is presented. A sample mixture containing several human growth hormone (hGH) isoforms was initially separated on a weak anion-exchange column. Fractions rich in 24 kDa hGH as determined by analytical SDS-PAGE were pooled and further separated by cation-exchange chromatography. The fractions pooled from the cation-exchange chromatography contained two hGH isoforms with a 2 kDa molecular mass difference according to SDS-PAGE analysis, 22 and 24 kDa hGH. The 22 and 24 kDa hGH were separated using continuous-elution preparative double-inverted gradient PAGE (PDG-PAGE) under nonreducing conditions. The preparative electrophoresis gel was composed of three stacked tubular polyacrylamide matrices, a 4% stacking gel, a 13-18% linear gradient gel, and a 15-10% linear inverted gradient gel. Fractions containing purified 24 kDa hGH were pooled and Western blot analysis displayed immunoreactivity to antihGH antibodies. PDG-PAGE provides researchers with an electrophoretic technique to preparatively purify proteins under nonreducing conditions with molecular mass differences of 2 kDa.

Chromatography↗
PubMed1999

Chromatographic separation of carotenoids.

The carotenoids are extremely reactive and consequently unstable due to their long system of conjugated double bonds. Several precautions, such as protection against light and oxygen, use of low temperature and antioxidants, analysis in the shortest possible time, should be taken during isolation and chromatography. The food samples, preferably fresh, are homogenized and immediately extracted with a suitable organic solvent. Saponification has been employed in order to hydrolyze the carotenoid esters, remove fatty material and destroy chlorophyll. This optional step facilitates subsequent carotenoid separation, identification and quantification. The separation of carotenoids is usually carried out by column chromatography, thin layer chromatography and high performance liquid chromatography, in analytical or preparative scale, on many stationary phases such as silica-gel, alumina, MgO, Ca(OH)2 and reversed-phase material (C18 and C30). The choice of the most suitable chromatographic method depends on the amount of sample, carotenoid composition, resolution, speed and purity required. Examples of carotenoid separation in different stationary phases will be shown and discussed.

Carotenoids↗